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Gene Therapy (2005) 12, 1353–1359 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt REVIEW Gene Therapy Progress and Prospects: Viral trafficking during infection EM Campbell and TJ Hope Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL, USA The intracellular steps involved in viral infection, namely cytoplasmic trafficking and nuclear import, are critical events in the viral life cycle that have lagged behind other areas of viral research. This review examines recent advances in our understanding of these steps for viruses commonly employed as viral gene delivery vectors. Steps governing the cytoplasmic trafficking and nuclear import of Herpes Simplex virus, Human Immunodeficiency virus and Adenovirus are reviewed in this article. Gene Therapy (2005) 12, 1353–1359. doi:10.1038/ sj.gt.3302585 Keywords: virus; infection; trafficking; HIV; HSV; adenovirus; cytoskeleton; nuclear import In brief Progress Prospects Cytoplasmic trafficking requires active transport due to properties of the cytoplasm that greatly impede diffusion. Most viruses overcome this barrier by engaging the transport machinery of the host cell microtubule cytoskeleton. The microtubule motor complex dynein is involved in the cytoplasmic transport of the viruses discussed. Nuclear import of viral genomes is an active process critical to infection or transduction of host cells. Identification of the viral components that mediate engagement and trafficking along microtubules will likely lead to more efficient vector systems. Some viruses may activate signal transduction pathways that augment their cytoplasmic trafficking, and identification and utilization of these pathways could aid in vector design. Elucidation of the mechanisms by which viruses facilitate the nuclear import of their genomes may generally enhance transduction and possibly allow for directed specific transduction of desired cell populations. Introduction Viral trafficking in the cytoplasm of target cells has for years been an area that lagged behind other areas of viral research. Studies involving viral fusion, gene regulation and viral production have predominated due to their relative amenability to classical biochemical and cell biology approaches. Models of viral infection have therefore concentrated on these events, often leaving readers with the impression that events between fusion and the virus reaching its replicative compartment, most frequently the nucleus, occur passively. However, it is now obvious that most virions utilize active transport to facilitate their nuclear localization. One fact often overlooked is that the environment of the cytoplasm is not something the size of a virion can diffuse through. Cytoskeletal structures and organelles present in the cytoplasm in fact create an environment Correspondence: Professor TJ Hope, Department of Microbiology and Immunology, University of Illinois at Chicago, 835 S. Wolcott Rm E-703, Chicago, IL 60612, USA that greatly hinders the diffusion of molecules and molecular complexes greater than 500 kDa. For example, when the density of the cytoplasm is taken into account, it has been estimated that it would take 231 years for a herpes virus to diffuse 10 mm in the axonal cytoplasm.1 The difficulties presented by limitations to diffusion in the cytoplasm have forced viruses to evolve methods to overcome this problem. It is becoming increasingly clear that the solution to this problem for most viruses is to utilize the cellular transport systems of the target cell cytoskeleton to promote their cytoplasmic trafficking. This review will discuss recent advances in our understanding of how different virions facilitate their cytoplasmic transport and nuclear translocation during infection, with specific emphasis on those viruses most utilized as gene delivery vectors. The previously mentioned gap between research examining viral trafficking during infection, and other areas of viral research, has been bridged in two ways. First, the use of pharmacological agents that disrupt cytoskeletal structures and dynamics have repeatedly demonstrated the importance of cytoskeletal-mediated Viral trafficking during infection EM Campbell and TJ Hope 1354 transport during infection for a large number of viruses (reviewed in Campell and Hope2 and Dohner and Sodeik1). These studies, however, must always be cautiously interpreted because of the fact that cytoskeletal disruption has pleiotropic effects on cell function. Therefore, experiments using such inhibitors must be viewed as a useful starting point used to design other, more definitive experiments. With that caveat, however, such agents continue to provide insights into trafficking processes that are otherwise very difficult to study. The second major advance allowing the examination of viral trafficking has been the use of fluorescent microscopy to visualize the behavior of individual fluorescently labeled virions in target cells. The ability to monitor the behavior of individual virions or groups of virions in target cells combined with selective fluorescent labeling of target cell components has been an invaluable tool in revealing how viruses interact with target cell components and compartments during infection. Again, as will be discussed in more detail below, these experiments have also indicated an intimate association of virions with the host cell cytoskeleton during infection. Cytoskeletal transport machinery is utilitized by viruses Utilization of cellular transport machinery has been a dominant theme in the examination of how viruses facilitate their intracellular trafficking. It is therefore important to briefly describe these systems, and we would also refer the reader to other, more comprehensive reviews.3,4 The maintenance of eukaryotic cellular organization is developed and maintained by the transport machinery of the cytoskeleton, which includes actin microfilaments, microtubules and intermediate filaments. Intermediate filaments are thought to primarily provide mechanical stability to cells and will therefore not be discussed further. The actin and microtubule cytoskeletons are formed from polymerization of monomeric subunits to form dynamic, three-dimensional structures in cells that provide the primary mechanisms of intracellular transport of molecular complexes greater than 500 kDa (reviewed in Dohner and Sodeik1). Both the microtubule and actin cytoskeleton form polarized filaments to which subunits are added to a growing, plus end in a complexly regulated process.5 These filaments then serve as the platforms that engage ATP-driven molecular motors to transport cargo throughout the cell (Figure 1). Transport along actin microfilamants is driven by myosin motors, with some myosins transporting cargo towards the plus end of filaments and others transporting cargo in the opposite direction. Another method of actin-based motility utilized by cells as well as intracellular pathogens, is the utilization of actin nucleating proteins to generate ‘actin comets’ that essentially propel cargo on the tip of a branched, rapidly polymerizing scaffold.5 While actin-based transport of cargo is thought to occur most frequently at the cell periphery and is, relatively speaking, used for short-ranged transport of cargo, longer-ranged transport is provided by the microtubule cytoskeleton (Figure 1). In addition to the polarity of individual filaments, microtubules also possess an organized, functional polarity in most cells whereby minus ends collectively end in a perinuclear Gene Therapy Figure 1 Cytoskeletal transport. The actin and microtubule cytoskeleton are responsible for trafficking numerous endogenous cargos, as well as viruses and intracellular bacteria, throughout the cell. In general, myosin motors facilitate short-range traffic of cargos in the cell periphery, whereas the dynein- and kinesin-microtubule-based motors transport cargos longer distances throughout the cell. Grey colored dots adjacent to cytoskeletal filaments represent myosin motors transporting cargo along microfilaments and dynein and kinesin motors transporting cargo along microtubules. area termed the microtubule-organizing center (MTOC) and plus ends extend into the cell periphery. Directional transport along microtubules is achieved by ATP-driven molecular motor complexes. In general, kinesins drive cargo towards the plus ends of microtubules extending to the cell periphery while dynein motor complexes drive cargo towards the minus ends located near the nucleus. Less is known about what determines the cargo specificity. However, a known critical player in the transport function of microtubules is dynactin (reviewed by Schroer6), which is a multiprotein dynein cofactor required for dynein function under many conditions. There is evidence that dynactin facilitates the interaction between cargo, particularly membrane bound cargo, and dynein.6 Disruption of the dynein complex with a dominant-negative protein known as dynamitin abolishes dynein-mediated traffic. However, the finding that dynactin disruption also abolishes the plus- and minus-end transport of some cargos that undergo bidirectional transport7 has led to the notion that dynactin may act to coordinate the net transport of bidirectionally transported cargo.3 Herpes simplex virus (HSV) trafficking and nuclear import HSV was one of the first viruses observed to associate with microtubules during infection, and these earlier Viral trafficking during infection EM Campbell and TJ Hope findings have been reviewed elsewhere.1,2 It is known that HSV utilizes microtubules to facilitate their translocation to a perinuclear region in the cell (Figure 2). Disrupting microtubules using pharmacological inhibitors prevents this accumulation of HSV in the perinuclear region.8,9 More recent investigations have focused on determining the molecular mechanisms by which HSV interacts with the microtubule transport machinery. As would be expected, given the minus-end directional transport HSV undergoes to reach the nucleus, numerous reports indicate that HSV interacts with dynein to facilitate its cytoplasmic transport.10,11 Using electron microscopy and immunogold antibodies, Sodeik et al8 demonstrated that dynein is present in the protein complex linking HSV virions to microtubules. Dohner et al10 have shown that HSV virions colocalize with the intermediate chain of the dynein protein complex as well as with the p150-glued protein of the dynactin complex, which is thought to link the dynactin complex to the dynein complex. Moreover, overexpression of dynamitin, to induce the disruption of the dynactin complex and inhibit dynein-mediated transport,6 prevents the nuclear accumulation of HSV virions in the nucleus and expression of early HSV gene products following infection.10 Taken together, these reports suggest dynein-mediated transport of HSV virions is a critical component of HSV intracellular trafficking. Other investigations have attempted to identify the HSV proteins responsible for interacting with the microtubule transport machinery. One HSV protein that is a viable candidate for providing the interaction between the incoming virion and the microtubule cytoskeleton is the VP22 tegument protein. The tegument is a virion compartment located between the capsid 1355 Figure 2 Microtubule transport of HIV and HSV. Both HIV (left panel) and HSV (right panel) have been observed to traffic in a microtubule-dependent fashion. Both viruses accumulate at the MTOC during infection and have been observed to tightly associate with microtubules during infection by fluorescent and electron microscopy. Reprinted with permission from McDonald et al21 and Sodeik et al.8 Gene Therapy Viral trafficking during infection EM Campbell and TJ Hope 1356 and envelope and may participate in postfusion events during HSV infection. Interestingly, VP22–GFP fusion proteins colocalize with microtubules and also affect microtubule dynamics, causing microtubule reorganization and stabilization when expressed in Vero cells (reviewed in Campbell and Hope2 and Dohner and Sodeik1). The ability of VP22 to bind microtubules has been mapped to a C-terminal domain and a central stretch of approximately 100 amino acids.12 VP22 may also play a role in facilitating the trafficking past the actin cortex, given the finding that VP22 also interacts with the actin motor myosin IIA.13 These facts make VP22 one of the most promising candidates for the mechanism by which incoming HSV virions associate with microtubules during infection. The herpes UL34 protein has also received attention as a protein that may be responsible for the interaction between HSV virions and microtubules during infection. UL34 interacts directly with the intermediate chain of dynein in yeast-two-hybrid models and glutathione Stransferase (GST) pull-down assays, and demonstrates perinuclear localization that is inhibited by nocadozole, a drug that disrupts the microtubule network.1,2 This notion is also supported by the fact that UL34 directly interacts with the viral capsid protein. However, UL34’s candidacy as a protein responsible for affecting virion trafficking during infection has recently been brought into question by the finding that extracellular virions do not contain detectable amounts of UL3414 and the fact that deletion of UL34 does not prevent infection or nuclear accumulation of HSV virions. Another protein recently proposed to mediate HSV retrograde transport during infection is the VP26 protein.11 Douglas et al11 demonstrated that VP26 binds the dynein light chains RP3 and Tctex1 in yeasttwo-hybrid systems and in vitro binding assays. They also found that microinjected HSV capsids lacking VP26 failed to accumulate around the nucleus as well as their VP26-negative counterparts.11 These observations make VP26 another attractive candidate to be the viral determinant that is responsible for interaction between HSV and the microtubule cytoskeleton during infection. Following cytoplasmic trafficking to the MTOC, HSV virions bind the nuclear pore complex (NPC) and release their DNA genome into the nucleus (reviewed by Whittaker15) (Figure 3). This process, reconstituted in vitro, has been shown to be inhibited with antibodies to the NPC complex or against importin b. In this sytem, HSV binding of the NPC was dependent on both importin b and Ran. It has also been found that uncoating of the HSV capsid and subsequent release of viral DNA requires both NPC binding and ATP. Given that the genome of HSV is, in viral terms, extremely large, a better understanding of the mechanisms by which HSV virions traffic within an infected cell will likely lead to more specialized vectors in which the genes not required for cellular transductions have been removed. As will be discussed in the next section, the removal of a few human immunodeficiency virus (HIV) accessory genes not required for transduction from HIVbased vectors resulted in the production of higher titer vectors from vector-producing cells. A similar if not more dramatic benefit could easily be imagined for HSV vectors as the HSV genes critical for gene transduction Gene Therapy Figure 3 Nuclear import of adenovirus and HSV. Electron micrographs showing the accumulation of (a) adenovirus and (b) HSV capsids at the nuclear envelope. Reprinted with permission from Saphire et al16 and Ojala et al.17 are identified and separated from the numerous other genes in its expansive genome. HIV trafficking and nuclear import Following fusion of the human immunodeficiency virus (HIV) envelope glycoprotein with CD4 and chemokine coreceptors at the cell surface, the viral core must traverse through the cytoplasm and induce the nuclear import of the viral nucleoprotein complex, known as the preintegration complex (PIC), and allow integration of the viral genome into the host cell chromosome. The characterization of these trafficking events, as well as the viral proteins that facilitate them, remains an active area of HIV research. The viral accessory protein Nef was one of the first viral proteins identified that appears to play a role in trafficking of virions in the cytoplasm of target cells. Nef has been shown to be incorporated into virions and increase virion infectivity (reviewed by Anderson Viral trafficking during infection EM Campbell and TJ Hope and Hope18). Importantly, numerous studies have found that Nef is no longer required to increase viral infectivity when virions are pseudotyped with pH-dependent envelopes, which are activated by the low pH of lysosomes and therefore enter the cell through an endocytic pathway. However, while the nature of the envelope affects the ability of Nef to increase viral infectivity, numerous studies agree that Nef does not affect the fusogenicity of HIV virions.2,19,20 This suggests that some property of these pH-dependent envelopes independent of their target receptor and fusogenic potential is responsible for their ability to complement the infectivity-enhancing properties of Nef. Importantly, these envelopes only fuse following the induction of endocytosis and trafficking of a virion within an endosome, some distance into the cell. A possible solution is that Nef acts to allow the virus to bypass the layer of cortical actin barrier present in cells. This would be consistent with the above data, as well as data demonstrating that Nef interacts with numerous molecules associated with cytoskeletal rearrangement, and our recent finding that disruption of the actin cytoskeleton in target cells increases the infectivity of Nef-deficient virions to wild-type levels.2 Our lab has also generated data demonstrating that the PIC utilizes microtubule-based transport to achieve a perinuclear translocation during infection21 (Figure 2). Utilizing fluorescent labeling, HIV virions have been observed to undergo bidirectional transport along microtubules that result in the accumulation of labeled virions in a perinuclear location. Moreover, viral reverse transcription complexes (RTCs), identified by the incorporation of fluorescent nucleotides, were observed to be associated with microtubules using correlative electron microscopy (Figure 2). The accumulation of RTCs at the MTOC appears to be dependent on the dynein motor complex, as microinjection of anti-dynein antibodies, known to disrupt dynein function, resulted in the prevention of this perinuclear accumulation of RTCs, and also, interestingly, resulted in these RTCs being transported to the periphery of the cell.21 This not only suggests that during infection, the RTC selectively interacts with the minus-end dynein motor complexes, but also that the RTC is capable of interacting with plusend kinesin motor complex, although this situation is clearly not predominant in the presence of active dynein motor function. Another element of HIV infection that may affect viral trafficking is the poorly described feature of viral uncoating. Uncoating involves the sequential loss of some viral structural proteins during infection (reviewed by Dvorin and Malim22). This is supported by a study examining PIC composition, which found that the p17MA protein is a component of purified PICs, but that the p24CA protein is not present. Immunofluorescent analysis of RTCs that have incorporated fluorescent nucleotides also showed that p17MA is present in most of these complexes, while many contained no detectable p24CA.3 It is tempting to speculate that some level of viral uncoating may, therefore, facilitate viral reverse transcription and/or trafficking events during infection, but currently there is only circumstantial evidence to support this. While many simple retroviruses require cell division and breakdown of the nuclear envelope to gain access to the host cell chromosome, lentiviral vectors such as HIV show considerable more promise than their retroviral counterparts because of their ability to transduce quiescent, nondividing cells (reviewed by Bartosch and Cosset23 and Cockrell and Kafri24). However, although there is clear agreement that this is the case, the mechanism that the viral PIC employs to traverse the nuclear membrane of target cells remains clouded. This confusion is due to the fact that a number of mechanisms have been reported to be the primary determinant allowing the PIC to achieve nuclear import. These have been reviewed extensively elsewhere.25,26 Briefly, convincing arguments have been made that the nuclear import activity of the PIC is mediated by the viral matrix, Vpr, integrase proteins and a DNA element formed during reverse transcription known as the central DNA flap. There remains a lack of consensus as to which, if any, of these elements are the primary determinant governing nuclear import of the PIC, and it may be that these elements act in a synergistic fashion in many settings. Identification of these proteins and nucleic acid structures involved in nuclear import are important for the design of recombinant HIV and other lentivirusbased gene transfer agents. For example, a vector packaging system lacking the Nef, Vif and Vpr genes of HIV has been shown to transduce growth arrested cells and monocyte-derived macrophages as efficiently, or better, than packaging sytems containing these genes (reviewed by Bartosch and Cosset23 and Cockrell and Kafri24), suggesting that, at least in the context of lentiviral gene delivery vectors, Vpr is not required for efficient transduction of nondividing cells. Although vector systems without the central polypurine tract (cPPT) that mediates the formation of the central DNA flap are known to efficiently transduce cells, various groups have shown that incorporation of the cPPT into HIV-based vectors can improve transduction efficiency of nondividing cells between two- and 10-fold.23,24 1357 Adenovirus trafficking and nuclear import It has been known for quite some time that adenovirus virions associate with microtubules. Adenoviruses are nonenveloped viruses that induce their own endocytosis by binding of its cellular receptor. Virions subsequently escape from the endosomal compartment via a mechanism that requires lowering of endosomal pH. Owing to space limitations, we confine our discussion to the cytoplasmic trafficking of adenovirus, but refer the reader to more comprehensive reviews examining adenovirus endocytosis and endosomal escape.27,28 Following entry into the cytosol, numerous studies have suggested that adenovirus utilizes microtubulebased transport to facilitate perinuclear accumulation. Observations that adenovirus associates with microtubules during infection and in vitro are almost 30 years old (reviewed in Campbell and Hope2). However, work in recent years has extended these observations to generate a much clearer picture of the events occurring during adenovirus infection. The first of a recent flurry of reports directly implicating microtubule-based transport during adenoviral infection came from Suomalainen et al. They found that Ad2 particles underwent bidirectional Gene Therapy Viral trafficking during infection EM Campbell and TJ Hope 1358 movement that resulted in a net displacement of virions towards the MTOC with peak velocities in the micrometer per second range.29 This transport was inhibited by microtubule depolymerization as well as by disrupting dynein function through dynamitin.29 Similar results were reported by Leopold et al examining the cytoplasmic trafficking of Cy3-labeled Ad5 virions. They also saw virions transported rapidly in curvilinear paths towards the nucleus in a fashion that was inhibited by microtubule depolymerization and microinjection of functionblocking antibodies directed at dynein, but not by actin depolymerization.30 However, this study found that Ad5 trafficking occurred almost exclusively in the minus-end direction,30 different from the bidirectional motility of Ad2 virions described by Suomalainen et al.29 Another report confirmed the net minus-end trafficking of adenovirus in enucleated cells.31 In an elegant series of experiments in which cell nuclei were removed from cells by centrifugation, Bailey et al31 demonstrated a stable interaction between adenovirus and the MTOC. This stable interaction, in addition to supporting the role of microtubules during adenovirus infection, also suggests that nuclear factors may be required for inducing the translocation of adenovirus from the MTOC to the nucleus during infection. Microtubule-directed motion has also been shown to occur during infection by adenoassociated virus.32 This study observed fluorescently labeled virions to undergo directed motion in curvilinear paths at speeds consistent with microtubule-based transport. Moreover, these authors observed individual virions traveling along identical curvilinear paths, suggesting transport of multiple virions on a single microtubule.32 The events facilitating microtubule-mediated nuclear transport of adenovirus have also been investigated. It has been reported that both the frequency and velocity of minus-end trafficking events during infection is increased by transient stimulation of the protein kinase A and p38 MAP kinase pathways by adenovirus.33 Despite the implication of these pathways, the precise mechanism by which this enhancement in trafficking occurs remains poorly defined. A recent report by Kelkar et al34 sought to determine the factors required for the adenovirus/microtubule interaction. Using an in vitro microtubule binding assay, these authors examined the binding of adenovirus to polymerized microtubules in the presence or absence of microtubule-associated proteins (MAPs), including dynein. They found that adenovirus bound microtubules strongly only in the presence of MAPs, and that the ability of MAPs to induce microtubule binding to adenovirus was greatly reduced when the MAP fraction was depleted of dynein.34 Following arrival at the nuclear membrane, adenovirus deposits its genome into the host cell nucleus (Figure 3). Studies examining the nuclear import of adenovirus throught the NPC have focused on identifying the cellular proteins involved in this process (reviewed in Greber and Fornerod35). It has been noted that nuclear import of adenovirus DNA can be inhibited by excess of other NLS-containing proteins, suggesting that adenovirus utilizes the classical nuclear import pathway to achieve nuclear localization of its genome. Moreover, the hsc70 protein is required for the nuclear translocation of viral DNA, but not the structural hexon protein. The role of the hsc70 protein in nuclear import is Gene Therapy not clear, however, and the authors speculate that hsc70 may play a role in uncoating of the virus following endosomal escape. They also suggest the alternative that VP22 may play a role conjugating an adenoviral NLS sequence to the nuclear import machinery, a role that hsc is thought to play in yeast. Another report has identified the CAN/Nup214 NPC protein as the docking site of Ad2 capsids, and found this association not to require additional cellular factors. These authors also find that the nuclear histone H1 protein is responsible for facilitating the uncoating of the adenoviral capsid at the NPC and subsequent translocation of the viral genome to the nucleus.35 As with the previous viruses mentioned, identification of the adenoviral gene products required for viral trafficking may allow not only for increased vector production and transduction efficiency, but may also limit the undesired immune responses generated at times by adenoviral vectors as the genes critical for transduction are dissected from those dispensable genes not required for gene transduction. Summary It is now clear that most viruses use components of the cytoskeleton to move within cells. As viruses are increasingly used as gene delivery vectors, an improved understanding of viral trafficking in the cytoplasm can only increase the efficiency of these vectors, as unnecessary viral gene products are separated from those required for gene transduction, potentially leading to increased vector production and transduction, and also possibly limiting undesired immune reactions in the host. Such optimization of the cell biology of gene therapy vectors will help bring the promise of gene therapy to reality. Acknowledgements We apologize to any authors whose work was not cited or cited indirectly due to space limitations. We thank Omar Perez, Marta Melar and Shivani K Maffi for critical review and discussions which improved the manuscript. References 1 Dohner K, Sodeik B. The role of the cytoskeleton during viral infection. Curr Top Microbiol Immunol 2005; 285: 67–108. 2 Campbell EM, Hope TJ. Role of the cytoskeleton in nuclear import. Adv Drug Deliv Rev 2003; 55: 761–771. 3 Welte MA. Bidirectional transport along microtubules. Curr Biol 2004; 14: R525–R537. 4 Karcher RL, Deacon SW, Gelfand VI. Motor-cargo interactions: the key to transport specificity. Trends Cell Biol 2002; 12: 21–27. 5 Pollard TD, Borisy GG. Cellular motility driven by assembly and disassembly of actin filaments. Cell 2003; 112: 453–465. 6 Schroer TA. Dynactin. Annu Rev Cell Dev Biol 2004; 20: 759–779. 7 Deacon SW et al. Dynactin is required for bidirectional organelle transport. J Cell Biol 2003; 160: 297–301. 8 Sodeik B, Ebersold MW, Helenius A. Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. J Cell Biol 1997; 136: 1007–1021. Viral trafficking during infection EM Campbell and TJ Hope 1359 9 Mabit H et al. Intact microtubules support adenovirus and herpes simplex virus infections. J Virol 2002; 76: 9962–9971. 10 Dohner K et al. Function of dynein and dynactin in herpes simplex virus capsid transport. Mol Biol Cell 2002; 13: 2795–2809. 11 Douglas MW et al. Herpes simplex virus type 1 capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and plays a role in retrograde cellular transport. J Biol Chem 2004; 279: 28522–28530. 12 Martin A, O’Hare P, McLauchlan J, Elliott G. Herpes simplex virus tegument protein VP22 contains overlapping domains for cytoplasmic localization, microtubule interaction, and chromatin binding. J Virol 2002; 76: 4961–4970. 13 van Leeuwen H, Elliott G, O’Hare P. Evidence of a role for nonmuscle myosin II in herpes simplex virus type 1 egress. J Virol 2002; 76: 3471–3481. 14 Reynolds AE, Wills EG, Roller RJ, Ryckman BJ, Baines JD. Ultrastructural localization of the herpes simplex virus type 1 UL31, UL34, and US3 proteins suggests specific roles in primary envelopment and egress of nucleocapsids. J Virol 2002; 76: 8939–8952. 15 Whittaker GR. Virus nuclear import. Adv Drug Deliv Rev 2003; 55: 733–747. 16 Saphire AC et al. Nuclear import of adenovirus DNA in vitro involves the nuclear protein import pathway and hsc70. J Biol Chem 2000; 275: 4298–4304. 17 Ojala PM et al. Herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro. Mol Cell Biol 2000; 20: 4922–4931. 18 Anderson JL, Hope TJ. Recent Insights into HIV Accessory Proteins. Curr Infect Dis Rep 2003; 5: 439–450. 19 Tobiume M, Lineberger JE, Lundquist CA, Miller MD, Aiken C. Nef does not affect the efficiency of human immunodeficiency virus type 1 fusion with target cells. J Virol 2003; 77: 10645–10650. 20 Cavrois M, Neidleman J, Yonemoto W, Fenard D, Greene WC. HIV-1 virion fusion assay: uncoating not required and no effect of Nef on fusion. Virology 2004; 328: 36–44. 21 McDonald D et al. Visualization of the intracellular behavior of HIV in living cells. J Cell Biol 2002; 159: 441–452. 22 Dvorin JD, Malim MH. Intracellular trafficking of HIV-1 cores: journey to the center of the cell. Curr Top Microbiol Immunol 2003; 281: 179–208. 23 Bartosch B, Cosset FL. Strategies for retargeted gene delivery using vectors derived from lentiviruses. Curr Gene Ther 2004; 4: 427–443. 24 Cockrell AS, Kafri T. HIV-1 vectors: fulfillment of expectations, further advancements, and still a way to go. Curr HIV Res 2003; 1: 419–439. 25 Piller SC, Caly L, Jans DA. Nuclear import of the pre-integration complex (PIC): the Achilles heel of HIV? Curr Drug Targets 2003; 4: 409–429. 26 Bukrinsky M. A hard way to the nucleus. Mol Med 2004; 10: 1–5. 27 Medina-Kauwe LK. Endocytosis of adenovirus and adenovirus capsid proteins. Adv Drug Deliv Rev 2003; 55: 1485–1496. 28 Meier O, Greber UF. Adenovirus endocytosis. J Gene Med 2003; 5: 451–462. 29 Suomalainen M et al. Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus. J Cell Biol 1999; 144: 657–672. 30 Leopold PL et al. Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis. Hum Gene Ther 2000; 11: 151–165. 31 Bailey CJ, Crystal RG, Leopold PL. Association of adenovirus with the microtubule organizing center. J Virol 2003; 77: 13275–13287. 32 Seisenberger G et al. Real-time single-molecule imaging of the infection pathway of an adeno-associated virus. Science 2001; 294: 1929–1932. 33 Suomalainen M, Nakano MY, Boucke K, Keller S, Greber UF. Adenovirus-activated PKA and p38/MAPK pathways boost microtubule-mediated nuclear targeting of virus. Embo J 2001; 20: 1310–1319. 34 Kelkar SA, Pfister KK, Crystal RG, Leopold PL. Cytoplasmic dynein mediates adenovirus binding to microtubules. J Virol 2004; 78: 10122–10132. 35 Greber UF, Fornerod M. Nuclear import in viral infections. Curr Top Microbiol Immunol 2005; 285: 109–138. Gene Therapy